Treatment of fluids, such as biological fluids for nutritional, cosmetic, diagnostic or therapeutic purposes, by irradiation is a widely investigated approach to inactivate microorganisms. Examples of irradiation treatment currently being studied include short-wave ultraviolet light, long-wave ultraviolet light or visible light with photosensitizing compounds, and broad-spectrum high-intensity flash light.
For instance, the food spoiling microorganisms such as Salmonella spp., Listeria monocytogenes, Mycobacteriae or the enterohemorrhagic Escherichia coli strain O157:H7 can readily contaminate fluids such as milk or fruit juices, fermented beverages, and processed beverages derived from them. Another example of contamination by microorganisms is the presence of protozoa, bacteria, and viruses in biological fluids, e.g. blood and its derivatives, or cell culture supernatants or lysates, from which medicines are obtained, which in turn may transmit these microorganisms to the recipient.
To inactivate these microorganisms, on a small laboratory scale, batch photoinactivation reactors have been developed. Such photoinactivation reactors are usually constructed as a cavity surrounded by an array of lamps. The sample is inserted into the cavity in a light-transparent container and exposed for a defined time. Additionally, the samples might be stirred to ensure homogeneous exposure. The cylindrical “Rayonet” and “Rayonette” reactors manufactured by the Southern New England Ultraviolet Company, Branford, Conn., and the light-chamber photoinactivation reactors manufactured by Luzchem Research Inc., Ottawa, ON, Canada, are typical examples of such photoinactivation reactors.
To ensure a thorough effective exposure of large volume of the fluid to be decontaminated, various flow-through reactors have been designed. These either spread the fluid to a thin film or a thin layer to minimize light intensity loss by self-absorption, or impose a transversal mixing on the longitudinally flowing fluid to effect an even illumination by transporting all volume fractions to the shallow illuminated outer liquid layer.
Caillet-Fauquet et al. (2004) describe a flow through UV-C irradiation method for inactivating bacteria and viruses using encephalomyocarditis virus spiked samples for biodosimetry or an in-house actinometry method for which no further details have been provided.
In the past, such aforementioned flow-through irradiation devices have been constructed as e.g., an inclined tube rotating axially around the tubular light source spreading the liquid on the inner wall into a thin free-flowing film (Habel and Sockrider 1947), a flat transparent cell squeezing the flow into a thin layer, or a transparent helical tube coiled around the tubular light source (Oppenheimer et al. 1959). In a flow-through device of a diameter exceeding the light penetration depth, transversal mixing can be generated either as Dean vortices in a coil, or by motionless mixers such as baffles in a tube, or by toroidal Taylor vortices between two countercurrent cylinders, where the liquid flows through the annular gap in-between.
If the light penetration is limited, the liquid can be spread into a thin film passing the light source, as described in U.S. Pat. Nos. 5,567,616 and 6,540,967. In liquid layers deeper than the light penetration depth, transversal mixing can in principle either be generated actively or passively.
A well-known principle for inducing active mixing is the generation of Taylor vortices between concentric countercurrent rotating cylinder surfaces. These Taylor vortices are superimposed on the longitudinal flow, the result of which is known as Taylor-Gortler flow. U.S. Pat. No. 6,576,201 discloses a cylindrical UV-C transparent flow-through cell with a static outer transparent wall and a turning inner cylinder. In a liquid layer in-between, the liquid pumped through the cell is mixed by counter-current Taylor vortices. A continuous-wave or a pulsed laser-UV source is disclosed for illumination.
Another device intended for active mixing is a flow-through-reactor based on the Archimedean screw, where the liquid pumped by the rotating screw is mixed in the coils (Della Contrada 2004).
The passive generation of mixing can either be achieved by turbulence, or by flow obstacles, such as motionless mixing baffles, in the flow path. Older designs of inactivation reactors are based on the flow of the fluid through an annular gap between the outer wall of the tubular lamp (or its envelope tube) and the inner wall of the concentric irradiation reactor. Such thin-layer irradiators are available commercially e.g. from Wedeco-Visa GmbH, Seewalchen, Austria.
Another effective technique of passive transversal mixing is the generation of Dean vortices in curved tubes, especially in helically coiled tubes. For instance, a coil reactor for the disinfection of highly absorbing fluids such as milk has been described, where a quartz glass coil is wrapped longitudinally around a plurality of low-pressure Hg vapor lamps (Bayha 1952). A similar coil reactor for the photosensitized inactivation of viruses in vaccines and other biological fluids has been built from borosilicate glass, where the coil is mounted around an incandescent filament lamp (Hiatt 1960). The coil is cooled in a light-transparent water jacket. Although the special craftsmanship and the fragility of borosilicate or quartz glass have limited the use of such tubular reactors in the past, the availability of UV-translucent, UV-resistant, and chemically inert fluoropolymers alleviates the design and scale-up of such flow-through-reactors. The coil is wrapped around a concentric tubular light source, usually a low-pressure mercury lamp, and transversal mixing is accomplished by the Dean vortices superimposing on the fluid flow. Such a helical envelope-reactor is described in the US Patent application 2003/0049809 A1. The dose received by the solution pumped through the reactor was calculated from the lamp intensity measurement by ferrioxalate actinometry, the absorbance, and the residence time (Wang et al. 2004).
There have been several attempts in the past to determine the energy of light effective during the irradiation. Light energy can be measured either by a photosensitive electronic sensor, or by a photochemical reaction effected by the light photons, or by its inactivation of an indicator microorganism present in or added to the biological fluid itself. Other theoretical methods comprise mathematical modeling simply by multiplying the residence time of a volume element with the intensity to calculate the dose, or in a more refined approach, by flow modeling to resolve residence time and dose.
Chemical actinometry measures the effect of light on a photochemical reactant mixture (Kuhn et al. 1989; Favaro 1998). In general, established photochemical actinometers with known quantum yield and temperature dependence can surpass electronic devices in reproducibility and stability. Photochemical reaction products should be measured on-line most conveniently e.g. by spectrophotometry or chemical sensors (Gauglitz 1983).
A preferred approach employs total opacity of the actinometric solution at the measured wavelength, so that the photochemical reaction occurs only at the very surface (Kuhn et al. 1989; Favaro 1998). Therefore sufficiently high reactant concentrations are preferred.
In a vaccine flow-through UV reactor for the inactivation of influenza viruses, the uridine actinometer was used to measure the lamp intensity (Zheleznova 1979).
Taylor-vortex generating devices have also been investigated as photochemical reactors for heterogeneous and homogeneous photochemical processes (Sczechowski et al. 1995, Forney and Pierson 2003). For the actinometric examination of the reactor, either the ferroixalate or the iodide/iodate actinometer was used. However, an absorbance-matching calibration to determine the dose effective in the iodide solution was neither done nor proposed (Forney and Pierson 2003).
For UVC measurement, the classical actinometers are uranyl oxalate (Bowen 1949,
Kuhn et al. 1989) and the ferrioxalate actinometer, but use of the first is limited by the Uranium radiotoxicity, and of the latter by UV-B, UV-A, and visible light sensitivity (Kirk and Namasivayam, 1983). Hydrogen sulfite or hydrogen cyanide adducts of triphenylmethane dyes also are as well known as UV-C-sensitive actinometers. For example, the colorless malachite green leucocyanide dissolved in ethanol does not show a long-wave UV or visible light sensitivity, but the green photo product absorbs additionally in the UV-C range (Calvert and Rechen 1952, Fisher et al. 1967). Azobenzene (Actinochrome 2R 245/440) in methanol enables a reuse of the actinometric solution (Gauglitz and Hubig 1981, 1984, 1985) as well as heterocoerdianthrone endoperoxide (Actinochrome 1R 248/334) (Brauer and Schmidt 1983). Despite the elegance of these complex organic compounds dissolved in organic solvents, it has been postulated that actinometric substances and solutions should be non-toxic and non-hazardous.
Immediately after various ultraviolet lamps became available in the beginning of the 20th century, acidic iodide solutions have been used as chemical actinometers (Bering and Meyer 1912). The low quantum yield of 0.05 lead to the use of nitrous oxide (N2O) as an electron scavenger (Dainton and Sills 1960, Rahn 1993). A more recent approach of opaque UV-C actinometry is the iodate-stabilized photodecomposition of iodide (Rahn 1997, Rahn et al. 1999, 2003). The triodide formed from iodide photolysis at 253.7 nm is determined spectrophotometrically at 352 nm or higher wavelengths (375 nm, 400 nm). This system has the advantage of insensitivity to wavelengths over 300 nm.
The use of an actinometer solution has been proposed to replace UV-sensors by flow-through or static probes containing the actinometer solution, which are inserted into the irradiation reactor. The concentrated actinometer solution, e.g. the iodide/iodate actinometer according to U.S. Pat. No. 6,596,542 or an uridine solution (Schulz et al. 2001) is pumped through a UV-transparent tube receiving the UV light from the lamp, or contained in a cell with a transparent window facing the UV lamp to be exposed for a defined time, and the photo products are then measured in a spectrophotometer. These sensors, however, measure only the fraction of radiation incident on them, but not the average fluence (light dose) effective on the fluid to be irradiated while contained within the irradiation reactor.
The use of a water-soluble triphenyl methane dye (4,4′,4″-tris-di-β-hydroxyethylaminotriphenyl acetonitrile) as an added actinometer substance is described for the “cold-sterilization” of microorganisms in fruit ciders, juices and plant saps by flow-through UV-C irradiation (Koutchma and Adhikari 2002). Equipment for this process is commercially available, e.g. the “CiderSure” or the “Sap Steady” thin-film irradiators manufactured by FPE Inc. of Macedon, N.Y., or the “Light Processed System” coiled tube irradiator manufactured by Salcor Inc., Fallbrook, Calif. Ciders and juices obtained from squeezed fruits show high turbidity from suspended particles, and high UV-C absorption from dissolved phenolic compounds and from ascorbic acid, and also a viscosity similar to protein solutions. Clarified apple juice has an absorption coefficient of 9/cm at 253.7 nm. The actinometer substance is added to the highly absorbing juice and irradiated in a collimated-beam apparatus, as used for the absolute UV inactivation kinetics determination of microorganisms in non-absorbing suspension (Bolton and Linden 2003). The absorption of the actinometer photoproduct at 600 nm increases, but in fact the added actinometer substance will only receive the light quanta fraction corresponding to its absorbance fraction of the total absorbance. The effective dose is then calculated from the destruction of the added actinometer dye. As it can be deduced from an “absorbed dose” of 190 mJ/cm2 inactivating no more than 3 log10 colony-forming units (cfu) E. coli K12/mL in apple juice in a petri dish, the addition of an actinometer substance to the sample itself delivers obviously false results for the effective dose. In non-absorbing suspension, 5 log10 cfu/mL E. coli are inactivated at ˜10 mJ/cm2 (Wright and Sakamoto 1999). However, the dose effective in the solution must be the same as the dose effective on the microorganism. The proposed addition of an actinometer substance to an already absorbing medium is therefore not capable to accomplish an exact effective dose measurement.
The biological dosimetry using a photoinactivatable microorganism was the first method to determine the applied dose in the irradiation of plasma, although details on the dosimetry using Aerobacter aerogenes (outdated name for Klebsiella pneumoniae) were not given. In addition, single-stranded DNA bacteriophages of the microviridae family such as S13 and Phi-X (phi chi) 174 give a linear decrease of titer with an increasing UV irradiation dose. Biodosimetry based on the inactivation of bacteriophages (e.g. Phi-X 174 or the single-stranded RNA bacteriophage MS2), or the inactivation of Bacillus subtilis spores, has been developed for testing flow-through ultraviolet water disinfectors. The dose-dependent un-attenuated inactivation rate of the bacteriophage Phi-X 174 in a dilute and UV-C transparent buffer suspension agitated horizontally in 33 mm petri dishes in the homogeneous radiation field of a 9 W UV-C lamp at an irradiance of 0.225 mW/cm2 was determined to be −0.44 (log10 pfu/mL)/(mJ/cm2) (Anderle et al. 2004).
The commercial thin-layer irradiator “CiderSure” (manufactured by FPE Inc., Rochester, N.Y.) used for fruit juices, essentially consists of an outer stainless-steel tube and an inner quartz tube, both in concentric and parallel mount to the centered tubular UV light source, where the fluid flows longitudinally through the annular gap in-between. It is validated by biodosimetry for every type of fruit juice and cider to adjust the flow-rate required for a >5 log10 colony forming units/mL reduction of E. coli O157:H7. Additionally, the light intensity loss over time is compensated by flow rate correction because the dose H is usually assumed as intensity E×residence time t (FDA 2000). However, this biodosimetric validation alone would not enable an optimization of the flow-through-reactor to achieve a narrow residence-time distribution thus avoiding excessive over-irradiation of the fluid. Even with nutritional fluids, this should be avoided, because UV over-dosage can generate an undesired off-flavor rendering the product unpalatable.
With all the aforementioned flow-through-devices, there are however rheological and technical limitations to overcome. Every volume element entering the irradiation zone is longitudinally dispersed into a faster flowing fraction, which receives a lower dose through its shorter residence time, and a slower tailing fraction, which receives a higher dose, and the volume fractions in between. FIG. 7 in U.S. Pat. No. 6,576,201 gives an example of a residence-time distribution dependent on the rotation rate of the inner cylinder in the Taylor vortex-generating cell. A dose too low can incompletely destroy the viable microorganisms and effect an incomplete inactivation. A dose too high can destroy the substances of interests, such as vitamins and flavors in juices, proteins in blood derivatives, or antigens in vaccines. Therefore it is desirable to optimize the irradiation process to make it sufficiently effective to destroy all target microorganisms and safe to preserve all substances of interest. A method to optimize such a process is therefore particularly desirable.
Another technical limitation of the aforementioned flow-through and batch reactor devices is the aging of the light sources used, which can lose a fraction of their initial light output during their operating lifetime. To compensate for that effect, an integrating counter has been used to ensure a constant and reproducible light dosage, however irrespective of the absorbance of the solution to be irradiated (Rideal and Roberts 1951). Moreover, the inactivation may be discontinued during operation due to a malfunctioning of a least one of the light sources. Although this decay and/or the switching off is detectable with an electronic light-sensitive sensor alone, the determination of its effect on the light dose effective on the fluid to be treated would require a measurement of such a dose to establish the relation between lamp intensity, absorbance, and dose decay, and to compensate such a decay by changing other process variables such as the flow rate. These devices also require re-validation at certain time intervals to ensure a consistent and effective operation. A method for measuring, controlling and compensating such fluctuations in light irradiation during light inactivation of microorganisms in a biological fluid, preferably in relationship to the absorbance of the biological fluid to be irradiated a batch reactor, and also a method for controlling that the inactivation process is carried out effectively despite fluctuations of the light irradiation are therefore particularly desirable.
Moreover, the light sources develop a considerable amount of thermal energy during operation of the irradiation devices such as batch-reactors and flow-through-reactors. The heating of the lamps in turn leads to considerable fluctuations of the light emission. In the low-pressure Hg vapor lamp, for example, the major part of the lamp power is converted into heat, and only around a third into light emission. Although low-pressure Hg vapor lamps seldom overheat beyond 60° C., temperature-sensitive substances of interest can suffer damage even by moderate heat. Direct cooling of only the flow conduit in the photoinactivation reactor will not thermostat the lamp and stabilize the intensity, and an adjustment of the lamp voltage to stabilize the intensity would not remove the heat, or the excessive heat. Accordingly, it would also be particularly desirable to develop flow-through-reactors which would be able to decrease the fluctuations of light irradiation caused by the heating of the light sources and which would preferably be able to keep the temperature of the lamp essentially constant or at least less fluctuating. Such a thermostatization has only been attempted with the 6-lamp centrifugal film irradiator as disclosed in U.S. Pat. No. 2,725,482 (Benesi 1956) where a water-cooled heatpipe for every lamp removed the excess heat. Other flow-through irradiators such as the Dill irradiator (U.S. Pat. No. 5,567,616), the baffled or other motionless mixer-elements containing tube type irradiator (U.S. Pat. No. 6,586,172), or the helical tube-type irradiator (WO 02/38191) have obviously been intended and disclosed only without a direct lamp thermostatization. In U.S. Pat. No. 6,586,172 assisted air-flow cooling (ventilation) is envisaged, which is however less effective than direct liquid thermostatization. The effectiveness depends on the ambient air temperature. In free-flowing thin-film irradiators the stream of coolant air may evaporate the water in the biological fluid. Liquid lamp thermostatization may ensure immediate operation at the maximum pathogenicidal intensity without burn-in time, as e.g. for low-pressure Hg vapor lamps with the maximum UV-C (253.7 nm) yield at 41.5° C., or may filter out infrared radiation, as e.g. from incandescent light sources, which would otherwise be absorbed and converted to excessive heat by the biological fluid.
In 1960, one of the leading experts in thin-film UV-C-irradiated vaccine technology stated: “In order to calculate the absolute quantity of energy involved in the virus inactivation itself, there must be some manner of quantitating the relative amounts of ultraviolet energy absorbed by the virus and the culture medium. This has not been possible as yet; furthermore the absolute exposure is dependent upon a number of variable factors: viscosity, temperature, surface tension, and frictional resistance of flow” (Taylor 1960). While such protein solutions or virus suspensions are usually clear or very slightly opalescent colloids, other fluids may constitute suspensions of filterable solids in a liquid. An investigation of the effect of various clay minerals on the inactivation of Klebsiella aerogenes bacteria in water has demonstrated a protection of the bacteria by UV-absorbing clays, but no such effect by UV-scattering clays (Bitton 1972). As recently shown for turbid apple cider and clear apple juice, the turbidity of such suspensions has the effect that at a similar apparent absorbance as measured in a spectrophotometer, microorganisms are inactivated faster in the turbid than in the clear fluid (Koutchma et al. 2004). The absolute exposure apparently depends also on the fraction of light scattered by the particles into the solution. Up until now no determination of the photochemically effective dose on microorganisms in a fluid sample has been reported in scientific or patent literature. Accordingly, a method for the determining the effective dose for inactivating microorganisms contained in a biological fluid, in particular a non-transparent biological fluid, preferably a method carried out in a flow through-reactor is desirable. Moreover, it is an object to provide a method for effectively inactivating microorganisms contained in a biological fluid, in particular a non-transparent biological fluid, preferably while leaving biologically active substances of interest unaffected.